There are now numerous examples of commercially valuable proteins which may be produced in large quantities by culturing a host organism capable of expressing heterologous genetic material. Once a protein has been produced by a host organism it is usually necessary to treat the host organism in some way, in order to obtain the desired protein. In some cases, such as in the production of interferon in Escherichia coli a lysis or permeabilisation treatment alone may be sufficient to afford satisfactory yields. However, some proteins are produced within a host organism in the form of insoluble protein aggregates which are not susceptible to extraction by lysis or permeabilisation treatment alone. It has been reported that a human insulin fusion protein produced in E. coli forms insoluble protein aggregates (see D. C. Williams et al (1982) Science 215 687-689).
A protein exists as a chain of amino acids linked by peptide bonds. In the normal biologically active form of a protein (hereinafter referred to as the native form) the chain is folded into a thermodynamically preferred three dimensional structure, the conformation of which is maintained by relatively weak interatomic forces such as hydrogen bonding, hydrophobic interactions and charge interactions. Covalent bonds between sulphur atoms may form intramolecular disulphide bridges in the polypeptide chain, as well as intermolecular disulphide bridges between separate polypeptide chains of multisubunit proteins, e.g. insulin. The insoluble proteins produced in some instances do not exhibit the functional activity of their natural counterparts and are therefore in general of little use as commercial products. The lack of functional activity may be due to a number of factors but it is likely that such proteins produced by transformed host organisms are formed in a conformation which differs from that of their native form. They may also possess unwanted intermolecular disulphide bonds not required for functional activity of the native protein in addition to intramolecular disulphide bonds. The altered three dimensional structure of such proteins not only leads to insolubility but also diminishes or abolishes the biological activity of the protein. It is not possible to predict whether a given protein expressed by a given host organism will be soluble or insoluble.
In our copending British Patent Application GB2100737A (an identical disclosure of which is contained in assignee's U.S. application Ser. No. 389,063, filed Jun. 16, 1982 now abandoned) we describe a process for the production of the proteolytic enzyme chymosin. The process involves cleaving a chymosin precursor protein produced by a host organism which has been transformed with a vector including a gene coding for the relevant protein. In the course of our work we discovered that the chymosin precursor proteins were not produced in their native form but as an insoluble aggregate. In order to produce a chymosin precursor in a native form which may be cleaved to form active native chymosin, the proteins produced by a host organism were solubilised and converted into their native form before the standard techniques of protein purification and cleavage could be applied.
In our copending published International Patent Application WO 83/04418 the methods used for the solubilisation of chymosin precursor proteins are described. In general the techniques described involve the denaturation of the protein followed by the removal of the denaturant thereby allowing renaturation of the protein. In one example the denaturant used is a compound such as urea or guanidine hydrochloride. When the insoluble precursor is treated with urea or guanidine hydrochloride it is solubilised. When the denaturant is removed, for example by dialysis, the protein returns to a thermodynamically stable conformation which, in the case of chymosin precursors, is a conformation capable of being converted to active chymosin.
The solubilised protein may be separated from insoluble cellular debris by centrifugation or filtration. The production of proteins from suitably transformed host organisms is potentially of great commercial value. The processes involved are of a type which may be scaled up from a laboratory scale to an industrial scale. However, where the protein produced is formed as an insoluble aggregate, potential complications in the process may increase the cost of production beyond a viable level. The solubilisation technique described above, whilst effective to solubilise such proteins, is relatively expensive and may represent a significant production cost.
We have discovered a generally applicable solubilisation process which, in its broadest aspect, does away with the requirement of relatively expensive reagents.